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Naturally-Occurring Entomopathogenic Fungi on Three Bark Beetle Species (Coleoptera: Curculionidae) in Bulgaria

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Naturally-Occurring Entomopathogenic Fungi on Three Bark Beetle Species (Coleoptera: Curculionidae) in Bulgaria Pestic. Phytomed. (Belgrade), 25(1), 2010, 59-63 UDC: 630*1:632.7:632.937.1:582.288(497.2) Pestic. fitomed. (Beograd), 25(1), 2010, 59-63 Scientific paper * Naučni rad DOI: 10.2298/PIF1001059D Naturally-Occurring Entomopathogenic Fungi on Three Bark Beetle Species (Coleoptera: Curculionidae) in Bulgaria Slavimira A. Draganova1, Danail I. Takov2 and Danail D. Doychev3 1Plant Protection Institute, 35 Panajot Volov Str., 2230 Kostinbrod, Bulgaria ([email protected]) 2Institute of Zoology, Bulgarian Academy of Sciences, 1 Tsar Osvoboditel Boulevard, 1000 Sofia, Bulgaria 3University of Forestry, 10 St. Kliment Ohridski Boulevard, 1756 Sofia, Bulgaria Received: December 3, 2009 Accepted: February 2, 2010 SUMMARY Bark beetles (Coleoptera: Curculionidae: Scolytinae) belong to one of the most damag- ing groups of forest insects and the activity of their natural enemies – pathogens, parasi- toids, parasites or predators suppressing their population density, is of great importance. Biodiversity of entomopathogenic fungi on bark beetles in Bulgaria has been investi- gated sporadically. The aim of this preliminary study was to find, identify and study mor- phological characteristics of fungal entomopathogens naturally-occurring in populations of three curculionid species – Ips sexdentatus Boern, Ips typographus (L.) and Dryocoetes autographus (Ratz.). Dead pest adults were found under the bark of Pinus sylvestris and Picea abies trees col- lected from forests in the Maleshevska and Vitosha Mountains. Fungal pathogens were isolated into pure cultures on SDAY (Sabouraud dextrose agar with yeast extract) and were identified based on morphological characteristics both on the host and in a culture. Morphological characteristics of the isolates were studied by phenotypic methods. The fungal isolates obtained from dead adults of Ips sexdentatus, Ips typographus and D. autographus were found to belong to the species Beauveria bassiana (Bals. – Criv.) Vuillemin, Beauveria brongniartii (Saccardo) Petch and Isaria farinosa (Holmsk.) Fries (anamorph Ascomycota, Sordariomycetes: Hypocreales, Cordycipitaceae). Morphological traits of the isolates are described. Keywords: Bark beetles; Ips sexdentatus; Ips typographus; Dryocoetes autographus; Entomopathogenic fungi; Beauveria bassiana; Beauveria brongniartii; Isaria farinosa; Morphological characteristics; Natural occurrence 59 Slavimira A. Draganova et al. INTRODUCTION of the northern range of Vitosha Mountain, West Bulgaria). Adults of Ips typographus were found under Ips typographus (L.) and Ips acuminatus Gyll. are the bark of dead spruce trees (Picea abies L.) in 2006. some of the most damaging bark beetles (Coleoptera: Adults of Ips sexdentatus were collected from the bark Curculionidae: Scolytinae) on living trees in Europe of dead Scots pine trees (Pinus sylvestris L.) in 2008 according to Gregoire and Evans (2004). from a second stand that was located at an altitude In Bulgaria, about 300 ha of spruce stands in the fo- of 1230 m above sea level on Maleshevska Mountain rest reserve Bistrishko branishte (Vitosha Mountain) (South-West Bulgaria). were destroyed some years ago due to damages caused Adults of D. autographus were found in 2008 under by Ips typographus (Georgiev, 2006). The activity of the bark of fallen and dead spruce trees in the forest re- their various natural enemies – pathogens, parasitoids, serve of Bistrishko Branishte (Vitosha Mountain). The parasites or predators suppressing their population den- stand was at 1550 m above sea level. sity is of great importance. Dead pest adults were examined for fungal pathogens While Beauveria bassiana (Bals.-Criv.) Vuillemin has in 2008 and 2009 at the Department of Biological and been reported as a fungal pathogen frequently occurring Integrated Pest Control (Plant Protection Institute). in populations of bark beetles, the species Isaria fumo- Cadavers of insects were placed in a moist chamber sorosea Wize (major synonym Paecilomyces fumosorose- to allow fungal reproductive structures to develop. us (Wize) Brown and Smith), Isaria farinosa (Holmsk.) Fungal pathogens were isolated into pure cultures on Fries (major synonym Paecilomyces farinosus (Holmsk. SDAY (Sabouraud dextrose agar with yeast extract) and ex Gray) Brown and Smith), Verticillium lecanii (Zimm.) were identified according to Samson et al. (1988) and Viegas and Metarhizium anisopliae (Metsch.) Sorokin Humber (1997) based on morphological characteris- have been mentioned only in a few reports (Landa et al., tics both on the host and in the culture. Morphological 2001; Jankevica, 2004; Sosnowska et al., 2004). characteristics of the isolates were studied by phenoty- Wegensteiner (2004), reviewing records about path- pic methods. Colonies growing for 14 days at 23oC un- ogens of bark beetles, pointed out that there was in- der daylight were checked on plates with PDA (potato sufficient knowledge of the activity of pathogens on dextrose agar) and SDAY. Conidia and conidiogenous bark beetles worldwide in spite of a presumed poten- cells were observed in smears and durable specimens tial of pathogens in controlling bark beetle popula- with lactophenol and aniline blue using a transmission tions. According to him, a number of investigations interference microscope BX60 DIC Olympus equipped have shown the occurrence of fungi in bark beetle with digital camera and Cell B image capture software field populations. Several fungal species were most fre- with a calibrated Carl Zeiss micrometer. quently mentioned, including B. bassiana and Isaria sp. (synonyms Spicaria, Paecilomyces). Biodiversity of entomopathogenic fungi on bark bee- RESULTS AND DISCUSSION tles in Bulgaria has been sporadically investigated. An isolate obtained from a dead adult of Ips sexdentatus It was established that the examined mycoses on Boer., collected from a natural environment, has been bark beetles were caused predominantly by the species identified by Draganova et al. (2007) as B. bassiana. Beauveria bassiana (Bals.-Criv.) Vuillemin. Two isolates The aim of this study was to find, identify and study (433 and 434 B. bassiana) from Ips typographus, three the morphological characteristics of fungal entomo- isolates (501, 502 and 503 B. bassiana) from Ips sexden- pathogens occurring naturally in populations of three tatus and one (559 B. bassiana) from D. autographus scolytid species – Ips sexdentatus, Ips typographus and were obtained. One isolate each of the species B. brong- Dryocoetes autographus (Ratz.). niartii (Saccardo) Petch (marked 435) and Isaria fari- nosa (Holmsk.) Fries (marked 500) were isolated in- to pure cultures from Ips typographus and Ips sexden- MATERIAL AND METHODS tatus, respectively. The three fungal species are an- amorphs of Ascomycota belonging to Sordariomycetes: Insects were collected from three localities. Hypocreales, Cordycipitaceae. The first stand was located at an altitude of 1400 m Dead insects placed in a moist chamber were cove- above sea level in a forest on Zlatnite Mostove (part red after three days by dense mycelium of the fungus 60 Pestic. fitomed. (Belgrade), 25(1), 2010, 59-63 Table 1. Size of conidia of Beauveria bassiana (Bals.-Criv.) Vuillemin isolates obtained from adults of bark beetles Size of conidia (μm) Fungal isolate Length Width Average size (minimum – maximum) (minimum – maximum) (Length x Width) 433 Beauveria bassiana 1.04-1.91 1.13-1.56 1.34 x 1.32 434 Beauveria bassiana 0.95-1.91 0.95-1.65 1.28 x 1.25 501 Beauveria bassiana 0.95-1.56 0.78-1.56 0.28 x 1.27 502 Beauveria bassiana 0.86-1.72 0.43-1.72 1.16 x 1.02 503 Beauveria bassiana 1.30-1.99 0.69-1.65 1.63 x 1.11 559 Beauveria bassiana 1.56-2.17 1.30-1.82 1.87 x 1.60 Table 2. Colony growth of the isolates of Beauveria bassiana (Bals.-Criv.) Vuillemin, Beauveria brongniartii (Saccardo) Petch and Isaria farinosa (Holmsk.) Fries on PDA (potato dextrose agar) and SDAY (Sabouraud dextrose agar with yeast extract) during 14 days of cultivation at 23oC Size of fungal colony on plates Size of fungal colony on Fungal isolate with PDA (mm) plates with SDAY (mm) Diameter Height Diameter Height 433 Beauveria bassiana 19.7 thin layer 21.3 3 434 Beauveria bassiana 17.7 thin layer 28.7 4 501 Beauveria bassiana 34.0 thin layer 20.3 2 502 Beauveria bassiana 27.3 thin layer 18.3 2 503 Beauveria bassiana 27.3 thin layer 20.7 2 559 Beauveria bassiana 21.7 thin layer 21.0 2 435 Beauveria brongniartii 20.7 thin layer 22.7 2 500 Isaria farinosa 34.0 thin layer 36.0 6 - white in the cases of B. bassiana and B. brongniartii Conidia of B. bassiana isolates (Table 1) were small- and yellowish white in the case of I. farinosa. Mycelium er in size than conidia of the species reported by Brady started growing out through the inter-segmental mem- (1979a) and by Evlakhova (1974) as 2-3 x 2-2.5 μm and brane of the host. as 2-3 μm in diameter, respectively, but they were sim- Conidiogenous cells of B. bassiana were densely clus- ilar in size to conidia of the isolate 426 B. bassiana tered in whorls, hyaline, smooth and short. In some with dimensions 1.8-2.0 μm in diameter (Draganova smears and durable specimens of all fungal isolates, it et al., 2007). was noticed that conidiogenous cells were branched, On SDAY, B. bassiana isolates formed round raised which made clusters thicker. Conidiogenous cells had colonies with powdery surface, with pigmentation from globose (isolates 433, 434, 503 and 559 B. bassiana) or white to cream, reverse of the colonies with pale cream flask-like base (501, 502 B. bassiana) terminating in pigmentation. Only the isolate 501 B. bassiana released narrow extended denticulate apex. The growing apex pink pigment into the media until the 10th day, but it repeatedly formed conidium and re-grew just below the paled and gradually disappeared and the redish reverse new conidium, giving a distinctly zig-zag appearance of the colonies became pink-tanned. On PDA the col- (rachis) which in some cases reached 12.86 μm (isola- onies were round, flat, like a hyaline film from radial te 501 B.
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